The present invention relates to a method for isolating DNA from biological samples such as blood, cells and biological tissues.
Transformation of microorganisms such as Escherichia coli and the like, the culturing of the resulting transformant, and the recovery of a desired plasmid DNA from the proliferating transformant have been carried out routinely in the field of genetic engineering. For the purpose of collecting DNA information concerning cancer and genetic diseases and using the DNA information for diagnosis, additionally, DNAs from biological samples such as blood, cells and biological tissues are recovered.
The method for more simply recovering and purifying plasmid DNAs from transformants includes, for example, methods described in Japanese Patent Laid-open No. 360686/1992, Japanese Patent Laid-open No. 23976/1996, R. Boom et al., J. Clin. MicroBiol. Vol. 28, No. 3, p. 495-503, and Japanese Patent Laid-open No. 250681/1995.
Among them, the chaotropic ion method described in R. Boom et al., J. Clin. MicroBiol. Vol. 28, No. 3, p. 495-503 is an excellent method for isolating DNA alone, comprising separating RNA and DNA comprised in microorganisms from each other by using DNA-absorbing carrier and chaotropic solution in combination. This method is also described by the Japanese Patent Laid-open No. 250681/1995 wherein the purification of only DNA from a microbial organism is carried out using two kinds of cartridges.
When the chaotropic ion method was used for biological samples such as blood, cells and biological tissues as subjects, DNAs from these biological samples could never be isolated without preliminary treatment of the biological samples, differing from the case of microorganisms. When carrying out the isolation of DNA from blood, proteins are also captured on the carrier by the chaotropic ion method, so that the DNA cannot be efficiently isolated or recovered.
A method for precipitating DNA comprised in blood in the presence of 0.5 to 0.6 M sodium chloride and a cationic surfactant alkylbenzyldimethylammonium salt is disclosed in U.S. Pat. No. 5,010,183.
Using this method, however, DNA cannot be recovered, when blood is used as the biological sample without any preliminary treatment (separation) or the like. The method requires preliminary treatment, for example leukocyte separation from blood. Furthermore, DNA yield or purity is not high.
It is an object of the present invention to provide a method for recovering purified DNA at a high DNA yield, using a biological sample without preliminary treatment.
It is an additional object of the invention to provide a method for recovering purified DNA at a high DNA yield, using a biological sample without preliminary treatment, wherein the method does not require complicated procedures such as centrifugation or extraction but requires the use of apparatuses of simpler structures and less procedures, so that the method can be automated. In view of the above, the method of the present invention does not require centrifugation between steps. For instance, the present invention does not require centrifugation between lysing a DNA-containing biological sample and forming a DNA bound carrier, which lysing and forming are discussed in more detail below. Similarly, the present invention does not require centrifugation between combining in a lysing solution a DNA-containing biological sample, a salt and a cationic surfactant, and supplying the lysing solution to a column with a DNA-binding carrier, which combining and supplying are discussed in more detail below. In view of this and other disclosure, the method of the present invention may be conducted without phosphate.
The invention relates to a method (first method) for isolating DNA from a biological sample, comprising
(a) putting a lysing solution, comprising a DNA-containing biological sample, a salt, and a cationic surfactant, and having a salt concentration the same or higher than the concentration initiating the inhibition of DNA precipitation (hereinafter referred to as precipitation inhibition-initiating concentration) into contact with a DNA-binding carrier to allow the DNA comprised in the biological sample to bind to the DNA-binding carrier;
(b) separating the DNA-bound carrier from other components;
(c) dissociating the bound DNA from the separated carrier; and
(d) recovering the dissociated DNA.
Furthermore, the invention relates to a method (second method) for isolating DNA from a biological sample, comprising
(A) supplying a solution, comprising a DNA-containing biological sample, a salt, and a cationic surfactant, and having a salt concentration the same or higher than the precipitation inhibition-initiating concentration, into a column with a DNA-binding carrier arranged on a membrane filter, said membrane having a solution retention potency and a solution permeation potency under aspiration, to allow the DNA in the biological sample to bind to the DNA-binding carrier;
(B) separating the DNA-bound carrier from other components, by removing the lysing solution under aspiration from the column;
(C) supplying a DNA-dissociating solution into the column, to dissociate DNA from the carrier; and
(D) recovering a solution comprising the dissociated DNA, by separating the dissociating solution under aspiration from the column.